Enyne acetogenins from Porcelia macrocarpa displayed anti-Trypanosoma cruzi activity and cause a reduction in the intracellular calcium level

Natural products are a promising source of new compounds with a wide spectrum of pharmacological properties, including antiprotozoal activities. Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is one of several neglected tropical diseases with reduced options for treatment, which presents limitations such as toxicity and ineffectiveness in the chronic stage of the disease. Aiming to investigate the Brazilian flora for the discovery of new anti-T. cruzi compounds, the MeOH extract from Porcelia macrocarpa R.E. Fries (Annonaceae) fruit peels displayed potent activity against trypomastigotes and intracellular amastigotes and was subjected to bioactivity-guided fractionation. Using different chromatographic steps, a fraction composed of a mixture of four new chemically related acetogenins was obtained. The compounds were characterized as (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-octadeca-13′,17′-dien-11′-inil)butanolide (1), (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-eicosa-13′,19′-dien-11′-inil)butanolide (2), (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-octadec-13′-en-11′-inil)butanolide (3), and (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-eicosa-13′-en-11′-inil)butanolide (4) by NMR analysis and UHPLC/ESI-HRMS data. The mixture of compounds 1–4, displayed an EC50 of 4.9 and 2.5 µg/mL against trypomastigote and amastigote forms of T. cruzi, respectively, similar to the standard drug benznidazole (EC50 of 4.8 and 1.4 µg/mL). Additionally, the mixture of compounds 1–4 displayed no mammalian toxicity for murine fibroblasts (CC50 > 200 µg/mL), resulting in a SI > 40.8 and > 83.3 against trypomastigotes and amastigotes, respectively. Based on these results, the mechanism of action of this bioactive fraction was investigated. After a short-time incubation with the trypomastigotes, no alterations in the cell membrane permeability were observed. However, it was verified a decrease in the intracellular calcium of the parasites, without significant pH variations of the acidocalcisomes. The intracellular damages were followed by an upregulation of the reactive oxygen species and ATP, but no depolarization effects were observed in the mitochondrial membrane potential. These data suggest that the mixture of compounds 1–4 caused an irreversible oxidative stress in the parasites, leading to death. If adequately studied, these acetogenins can open new insights for the discovery of new routes of death in T. cruzi.


Scientific Reports
| (2023) 13:10254 | https://doi.org/10.1038/s41598-023-37520-3 www.nature.com/scientificreports/ million people worldwide, an estimated seven million are affected in Latin America where being about one million women of reproductive age 1, 10 . It is assumed that CD causes about US$ 7 billion/year in production loss, cardiovascular disease treatment, and health care worldwide within US$ 1 billion only in South America 5,6 . Due to a low investment, there are only two drugs available for the clinical treatment, benznidazole and nifurtimox, with efficacy restricted to the acute phase and intense side effects 10,11 . Since cardiac and gastro-intestinal patients are especially susceptive to COVID-19 complications, the outbreak increases the urgency for new drugs and treatments, especially due to impacted health services, and worsening prognostics [12][13][14][15] . In this context, natural products are an alternative in the prospection of new drug prototypes 16 . Previous studies from our research group with Porcelia macrocarpa R.E. Fries (Annonaceae) led to the isolation of metabolites with in vitro potential against trypomastigote and amastigote forms of T. cruzi. As reported 17,18 , extracts from seeds and flowers afforded acetylene acetogenins and fatty acids with potent activity against trypomastigotes forms of T. cruzi caused by depolarization of both the plasma membrane and mitochondrial membrane potential of the parasite. As part of our continuous studies with this plant, the MeOH extract from fruits peels of P. macrocarpa was prepared and the effect against trypomastigotes and amastigotes of T. cruzi was investigated. As a result, it was observed that this extract caused 100% of parasite death at 300 µg/mL indicating the presence of bioactive metabolites. Using a bioactivity-guided approach, it was obtained a fraction composed of a mixture of four new enyne acetogenins (1-4) which were chemically characterized by NMR and UHPLC/ESI-HRMS and evaluated against T. cruzi trypomastigotes and amastigotes. Finally, insight into the mechanism of lethal action of the mixture composed by 1-4 was investigated.
Anti-T. cruzi activity. The mixture of compounds 1-4 (fraction D-1) displayed EC 50 values of 4.9 and 2.5 µg/mL against trypomastigotes and intracellular amastigotes, respectively (  18 evaluated the effect of related acetogenins from seeds of P. macrocarpa and compounds with same γ-lactone unity, but containing a side chain with a triple bond at C-11′ and one double bond at C-19′ displayed moderate activity against amastigotes. Otherwise, no effects against T. cruzi were observed when a saturated side chain was present, indicating that unsaturation is a crucial feature for the antiparasitic activity. As observed in this work, the effect of the mixture of acetogenins 1-4 against T. cruzi was quite similar to the standard drug benznidazole. This data demonstrates that the presence of the enyne conjugated system plays an important role in the potential against the parasite. Therefore, due to the high activity and improvement of SI when compared to benznidazole, the fraction composed by 1-4 was subjected to further biological studies to evaluate its mechanism of action against trypomastigotes of T. cruzi.
Mechanisms of action studies. The lethal mechanisms of the fraction D-1, containing compounds 1-4, was performed to evaluate the plasma membrane permeability, the analysis of intracellular calcium levels, the acidocalcisome alkalinization and mitochondrial membrane alterations, including the electric potential, reactive oxygen species and the ATP levels.
Plasma membrane permeability. The plasma membrane regulates transport of ions and nutrients as well as pH and other factors 21 . As such, alterations in its composition can modify the fluidity and contribute to trypomastigote death 22 . Amphotericin B, a clinical drug used in the treatment of Leishmania spp., causes permeabilization of the plasma membrane of this protozoan parasite 23 . To evaluate this effect we incubated the mixture of compounds 1-4 (fraction D-1) with trypomastigotes for up to 120 min at the EC 50 value of 4.9 µg/mL and detected alterations in the fluorescence levels using Sytox Green, a fluorescent probe that binds to nucleic acids and increases the fluorescence after membrane damages 24 . Triton X-100, a non-ionic detergent, was used as a positive control to induce the solubilization of the lipid bilayer 25 . No increases in fluorescence levels were observed after treatment with the mixture of compounds 1-4 ( Fig. 2), suggesting no effect in the membrane of the parasite.
Intracellular Ca 2+ levels. Calcium ions regulate several signalization pathways, parasite differentiation, flagellar function and are essential for trypanosomatids survival. Alteration of calcium homeostasis can result in the parasite death 26,27 . Using the fluorescent probe Fluo-4AM dye, it was observed that the mixture of compounds 1-4 (fraction D-1) caused reduction of cytosolic Ca 2+ levels at all evaluated times of the incubation (Fig. 3), suggesting a metabolic imbalance. To evaluate the intracellular calcium, a known organelle in trypanosomatids was studied, named acidocalcisomes.
Acidocalcisome alkalinization. Acidocalcisomes are acidic organelles and the main Ca 2+ storage for trypanosomatids, where the ion is bound to polyphosphates 27,28 . The alkalinization of these organelles has been related to leakage of calcium to the cytoplasm 26 . No effect was observed after treatment with the mixture of compounds 1-4 Table 1. Anti-T. cruzi activity and cytotoxicity of mixture of acetogenins 1-4 (fraction D-1) from P. macrocarpa fruit peels and positive control benznidazol. a EC 50 , 50% effective concentration; b CC 50 , 50% cytotoxic concentration; c SI, selectivity index (CC 50 mammalian cells/EC 50 parasite form). www.nature.com/scientificreports/ (fraction D-1- Fig. 4), corroborating the reduction of calcium levels in the cytoplasm as previously observed.
After treatment with the mixture of compounds 1-4, the decrease of the calcium levels in the cytoplasmic milieu of the parasite may be a result of the influx of this ion to the mitochondria or the endoplasmic reticulum, since both organelles are natural reservoirs of calcium in the parasite 27,28 . Considering that an elevated concentration of calcium can harm the mitochondria 29 , the electric potential of this vital organelle was further investigated.
Mitochondrial membrane potential (Δψm). The single mitochondrion is unique to the trypanosomatids and is essential for the bioenergetic metabolism. It is also involved in calcium homeostasis as a known source of reactive oxygen species (ROS). Due to these characteristics, the T. cruzi mitochondria has been considered an interesting target for new anti-trypanosomal drugs 29,30 . Compounds that affect the respiratory chain tend to impact the ROS production and can also induce oxidative stress. Using the JC-1 probe, alterations in the mitochondrial membrane potential were evaluated. As result, no decreases of the fluorescence levels were detected after treatment with the mixture of compounds 1-4 (fraction D-1- Fig. 5), when compared to untreated parasites. This data reflects no direct impact of the compounds to the organelle. It has been shown that the overloading of calcium by the mitochondria can affect the mitochondrial membrane potential 31 , but this effect was not observed after treatment with the mixture of compounds 1-4. Although no electric alterations were detected in the mitochondria, alterations of reactive oxygen species levels were studied.     www.nature.com/scientificreports/ Reactive oxygen species levels (ROS). The levels of ROS were also investigated after treatment with the mixture of compounds 1-4 (fraction D-1). Using the fluorescent probe H 2 DCFDA, a significant (p < 0.05) increase in ROS levels was observed after 2 h of incubation (Fig. 6). Despite no statistical differences, the ROS levels were clearly increased after 1 h incubation when compared to the untreated parasites. Besides the cellular membrane, endoplasmic reticulum and the cytoplasm, the mitochondria contribute to 90% of the ROS production. The imbalance between mitochondrial reactive oxygen species and removal due to overproduction of ROS and/ or decreased antioxidants defense activity results can cause the oxidative stress. These molecules can damage several cellular constituents as DNA, proteins and lipids 32 . Additionally, the detoxification of ROS in Trypanosomatids are done by thiols and the enzyme trypanothione reductase. When these systems fail to cope with an excessive production, these highly reactive molecules (ROS) can induce an oxidative stress, leading to a cellular death 32 . Due to this toxic effect after treatment with the mixture of compounds 1-4, we investigated the ATP levels.
ATP levels. Considering the increased levels of ROS and the possible damages caused by these highly reactive compounds, we investigated the ATP levels of the parasite using a luminescent assay with luciferin. The mixture of compounds 1-4 (fraction D-1) caused a significant increase in ATP levels after 1 h of treatment (Fig. 7) when compared to untreated cells. In parallel, ROS upregulation was also observed after 1 h of incubation as previously discussed (Fig. 6). Increased levels of ROS have been accompanied by elevated release of ATP 33 . Mitochondrial oxidative phosphorylation is the major ATP synthetic pathway in eukaryotes, including Trypanosomatids. During this process, the reduction of O 2 molecules produces several reactive oxygen species (ROS) 34 . It has also been known that Ca 2+ is a physiological stimulus for ATP synthesis and consequently, a stimulus for ROS generation 34 .
Considering the tight connection among these metabolic compounds, the alteration of any of them can naturally affect the production of the other. In our studies, it is possible that the increased levels of ATP can be a cellular effort to fight the oxidative stress caused by the treatment with the mixture of compounds 1-4.

Conclusions
Using a bioactivity-guided approach, four new chemically related acetogenins 1-4 were characterized from the fraction D-1 obtained from the MeOH extract of P. macrocarpa fruit peels by NMR and UHPLC/ESI-HRMS analysis. The mixture of compounds 1-4 displayed anti-trypanosomal activity against trypomastigote and amastigote forms of the parasite with a similar effect to the standard drug benznidazole. Additionally, these new compounds showed no cytotoxicity against NCTC cells, demonstrating a promising potential. Due to this activity, mechanism of action studies were performed. The fraction containing compounds 1-4 reduced the cytosolic calcium levels, with no alkalinization of the acidocalcisomes. Although it induced no depolarization of the mitochondrial membrane potential, increased levels of ATP were detected, as a possible cellular effect to counterbalance the oxidative stress caused by the elevated ROS levels leading to the parasite death.  Extraction and bioactivity-guided fractionation. The fruit peels were removed immediately after collection and dried at room temperature. After milling, the plant material (168 g) was defatted (10 × 1L) with n-hexane. The residual material was then extracted with MeOH (8 × 1L) at room temperature. After elimination of the solvent under reduced pressure, was obtained the MeOH extract (35 g) which was resuspended in MeOH:H 2 O 1:1 and successively partitioned using n-hexane and EtOAc. As both crude MeOH extract and n-hexane phase displayed activity against T. cruzi (100% of parasite death at 300 µg/mL), part of the n-hexane phase ( Parasite and mammalian cell maintenance. Trypomastigotes (Y strain) were maintained in rhesus monkey kidney cells (LLC-MK2-ATCC CCL 7) using RPMI-1640 medium supplemented with 2% bovine fetal serum (BFS) in an incubator humidified with 5% CO 2 at 37 °C. LLC-MK2 and conjunctive mice cells (NCTC clone L929, ATCC) were maintained at 37 °C with RPMI-1640 supplemented with 10% fetal bovine serum at 5% CO 2 . Macrophages were obtained from the peritoneal cavities of BALB/c mice through washing with the same medium 36,37 .

Methodology
In vitro activity against trypomastigote forms of T. cruzi. Trypomastigotes were obtained from previously infected LLC-MK2 cultures and counted in a Neubauer hemocytometer seeded at 1 × 10 6 cells/well (96 well plates). The mixture of compounds 1-4 (fraction D-1) was added in serial dilution (100-1.6 µg/mL) using RPMI-1640 medium and the plated were incubated for 24 h at 37 °C in a 5% CO 2 -humidified incubator. Benznidazole was used as the standard drug. Trypomastigote viability was evaluated by the resazurin assay 17,36 .
In vitro activity against amastigote forms of T. cruzi. Previously obtained macrophages were plated on 16 well chamber slides (NUNC®, Merck) at 1 × 10 5 macrophage/well and incubated for 24 h at 37 °C in a 5% CO 2 -humidified incubator. Trypomastigotes were washed with RPMI-1640 medium and used to infect the cells at 1 × 10 6 /well for 2 h. After this time, non-internalized parasites were removed by washing with RPMI-1640 medium. Serial dilutions (100-1.6 µg/mL) of the mixture of compounds 1-4 (fraction D-1) were incubated with infected macrophages for 48 h at 37 °C and 5% CO 2 with benznidazole as the standard drug. Slides were then fixed with MeOH and stained with Giemsa. EC 50 values were obtained after counting infected cells with an light microscope (EVOS M5000) and calculated according to literature 35,37 .
Mechanism of action studies. SYTOX Green assay for cell membrane permeability. Washed trypomastigotes (2 × 10 6 /well) were incubated with 1 µM SYTOX Green probe (Molecular Probes) in HANKS' balanced salt solution (HBSS, Sigma-Aldrich) supplemented with 10 mM D-Glucose in the dark. The mixture of compounds 1-4 (fraction D-1) was added (t = 0 min) at the EC 50 value of 4.9 µg/mL and the fluorescence levels were measured every 20 min for 120 min. Untreated parasites were the negative control and maximum permeabilization was achieved with 0.5% Triton X-100. Fluorescence intensity was determined by fluorometric microplate reader (FilterMax F5) with excitation and emission wavelengths of 485 and 520 nm, respectively. Triton X-100 was added to all samples at the end of the assay. www.nature.com/scientificreports/ washed and treated with the mixture of compounds 1-4 (fraction D-1) at the EC 50 value of 4.9 µg/mL. Using a fluorometric microplate reader (FilterMax F5 Multi-mode, Molecular Devices), fluorescence levels were measured for 260 min (485 and 535 nm for excitation and emission wavelengths, respectively). Negative control consisted of untreated parasites and maximum intracellular calcium levels were obtained using 0.5% Triton X-100 (v/v) 38,39 .

Measurement of intracellular calcium (Ca
Alterations in acidocalcisomes. Trypomastigotes (2 × 10 6 /well in 96 well plates) were washed with PBS and incubated with acridine orange dye (Thermo Fisher Scientific) at 4 µM for 5 min in the dark. Non-internalized probe was removed with PBS. The mixture of compounds 1-4 (fraction D-1) was incubated with the parasite at the EC 50 value of 4.9 µg/mL for 120 min. Fluorescence levels were measured using Multimode FilterMax F5 (Molecular Devices) microplate reader with excitation and emission wavelengths of 485 and 535 nm, respectively. Nigericin (4 µM) was used as positive control and non-treated parasites as negative control 39 .
Mitochondrial membrane potential (Δψm). Trypomastigotes (2 × 10 6 /tube) were washed with HBSS supplemented with NaHCO 3 and D-glucose and incubated with the mixture of compounds 1-4 (fraction D-1) at the EC 50 value of 4.9 µg/mL, at 37 °C and 5% CO 2 for 1 and 2 h. After washing, JC-1 dye (10 µM) was added and the parasites were incubated for 20 min at 37 °C in the dark. Fluorescence was measured in a Attune NxTflow cytometer (488 and 530/574 nm excitation and emission wavelengths, respectively). The mitochondrial membrane potential was determined by the ratio between red/green fluorescence intensities (BL-2/BL-1). Positive and negative control were CCCP (Sigma-Aldrich) at 100 µM and non-treated parasites, respectively 39 .
Measurement of ROS levels. The mixture of compounds 1-4 (fraction D-1) was incubated at the EC 50 value of 4.9 µg/mL with washed trypomastigotes (2 × 10 6 /well) in black 96 well plates in HBSS supplemented with NaHCO 3 (4.2 mM) and D-glucose (10 mM), at EC 50 value for 1 and 2 h (24 °C, 5% CO 2 ). Azide (10 µM) and untreated parasites were used as positive and negative controls, respectively. Afterwards, H 2 DCFDA (20 µM) was incubated with the parasites for 15 min at 37 °C. Fluorescence intensity was measured using FilterMax F5 microplate reader (485 and 520 nm excitation and emission wavelengths, respectively). Controls containing only the compound and the fluorophore were added in all experiments to measure interference 40,41 .
Measurement of ATP levels. The mixture of compounds 1-4 (fraction D-1) was incubated at the EC 50 value of 4.9 µg/mL with trypomastigotes (2 × 10 6 /well) in HBSS and glucose for 1 h, at 37 °C. Parasites were lysed with 0.5% Triton X-100 and mixed with a standard reaction buffer (ATP Determination kit-Thermo Fisher) containing DTT (1 mM), luciferin (0.5 mM) and luciferase (1.25 µg/mL). A luminometer (FilterMax F5 Multi-mode, Molecular Devices) was used for measuring luminescence intensity and ATP quantity calculated with an ATP standard curve 39 .
Statistical analysis. Samples were tested in duplicates of three independent assays. Calculation of EC 50 and CC 50 values was performed using dose-response sigmoid curves in Graph-Pad Prism (GraphPad Prism software 6.0, San Diego, CA, USA). Mechanism of active studies were analyzed using ANOVA turkey test for significance (p < 0.05) 38

Data availability
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